The value of bone marrow examination for tumor staging in breast cancer

Summary The purpose of our study was to investigate the value of cytokeratin antibodies for identifying bone marrow involvement in breast cancer patients who showed no evidence of distant metastases using noninvasive tumor staging procedures. Bone marrow for histological (biopsy) and immunocytochemical (aspiration) evaluation was obtained from the anterior iliac crest from 50 unselected consecutive women during surgical treatment of the primary tumor. The histological examination was done on nondecalcifed bone sections. The immunocytochemical studies were carried out on interface smears of the bone marrow aspirates. For staining, cytokeratin antibodies (PKK 1) and the immune alkaline phosphatase method was used. Cytokeratin-positive cells were found in 4 of the 50 cases (8%). Of those 4 patients, however, 2 also showed evidence of neoplastic bone marrow infiltration histologically. We thus were able to prove that immunocytochemistry on aspirates is superior to conventional histology in identifying tumor in bone marrow. Nonetheless, our results clearly fell below the rate found in previous studies where epithelial membrane antigen antibodies were used.     

Heterogeneous proliferative potential of occult metastatic cells in bone marrow of patients with solid epithelial tumors

Bone marrow is a major homing site for circulating epithelial tumor cells. The present study was aimed to assess the proliferative capacity of occult metastatic cells in bone marrow of patients with operable solid tumors especially with regard to their clinical outcome. We obtained bone marrow aspirates from 153 patients with carcinomas of the prostate (n = 46), breast (n = 45), colon (n = 33), and kidney (n = 29). Most of the patients (87%) had primary disease with no clinical signs of overt metastases [tumor-node-metastasis (TNM)-stage UICC (Union Internationale Contre le Cancer) I-III]. After bone marrow was cultured for 21-102 days under special cell culture conditions, viable epithelial cells were detected by cytokeratin staining in 124 patients (81%). The cultured epithelial cells harbored Ki-ras2 mutations and numerical chromosomal aberrations. The highest median number of expanded tumor cells was observed in prostate cancer (2,619 per flask). There was a significant positive correlation between the number of expanded tumor cells and the UICC-stage of the patients (P = 0.03) or the presence of overt metastases (P = 0.04). Moreover, a strong expansion of tumor cells was correlated to an increased rate of cancer-related deaths (P = 0.007) and a reduced survival of the patients (P = 0.006). In conclusion, the majority of cancer patients have viable tumor cells in their bone marrow at primary tumor diagnosis, and the proliferative potential of these cells determines the clinical outcome.     

A monoclonal antibody cocktail for detection of micrometastatic tumor cells in the bone marrow of breast cancer patients

We investigated whether monoclonal antibodies (MoAbs) reactive against both acidic and basic cytokeratins alone were sufficient to detect minimal numbers of contaminating epithelial tumor cells in the bone marrow of breast cancer patients. Monoclonal anti-cytokeratin antibodies (AE1 and AE3) were used to stain 14 breast carcinomas by the avidin-biotin-peroxidase technique. Nine tumors (64.3%) showed high reactivity and five (35.7%) showed low or moderate reactivity. Nine MoAbs that proved to be unreactive to light density bone marrow cells by immunoalkaline phosphatase histochemistry were screened for reactivity to breast carcinomas having only low or moderate positivity to cytokeratin antibodies. Three of nine MoAbs showed high percentages of positivity and were selected to supplement the anti-cytokeratin antibodies for immunohistochemical detection of minimal marrow disease in breast cancer patients. A MoAb cocktail was prepared, further tested for reactivity to another five breast carcinomas, and compared with cytokeratin staining alone. The cocktail labeled 100% of carcinoma cells in all the examined specimens. To determine the sensitivity of this panel for detecting minimal numbers of contaminating tumor cells in bone marrow, in vitro mixing experiments were performed. T47D breast carcinoma cells were mixed with bone marrow mononuclear cells at ratios from one tumor cell per 10 bone marrow cells up to one tumor cell per 1 x 10(6) marrow cells, and cytospin preparations were subsequently stained with the MoAb cocktail by the immunoalkaline phosphatase method. Our approach could detect one tumor cell in 1 x 10(5) hematopoietic cells.     

Quantitation of the monoclonal plasma cell component in bone marrow from patients with serum paraproteinemia and nondiagnostic marrow morphology

Twenty-one patients with serum monoclonal gammopathy but lacking acceptable morphological evidence of myelomatosis were studied with reference to the degree, if any, of monoclonal plasma cell expansion in aspirated marrow samples, enriched for plasma cells and analysed with respect to light chain distribution. Four of these patients had a biopsy-proven plasmacytoma of bone. Bone marrow aspirated from sites distant to the tumor showed clear evidence of infiltration by monoclonal plasma cells in two of the cases studied; the other two patients had normal results. Of the 17 other cases, 14 showed evidence of a monoclonal plasma cell component qualitatively concordant with the serum paraprotein as one would expect. These cases could be subdivided into those with myeloma (six cases) and those with monoclonal gammopathy of undetermined significance (eight cases) on the basis of conventional biochemical and radiological criteria. Three of the 17 patients, however, did not show evidence of monoclonal plasma cell infiltration, despite the presence of lytic lesions. It is important to recognize this minority group that simulates myeloma but that may well reflect alternative pathology that has not been identified.     

Complement depletion in vitro limits monoclonal antibody 6-19-dependent complement-mediated killing of tumor cells in bone marrow

Selective killing of nonhematopoietic tumor cells in bone marrow harvested for autologous bone marrow transplantation was studied using cultured neuroblastoma cells, monoclonal antibody 6-19, and baby rabbit serum as a source of complement. Monoclonal antibody 6-19, a murine IgG2a antibody, was selected for binding to a cell surface antigen on cultured human neuroblastoma cells that is not expressed by hematopoietic cells. The antigen is an 80-kd sialoglycoprotein present on a wide variety of nonhematopoietic tumors, and it is expressed by normal fibroblast and endothelial cells. The effect of the presence or absence of bone marrow mononuclear cells on target cell survival was evaluated using Hoechst 33342-stained neuroblastoma cells, trypan blue exclusion, and fluorescence microscopy. More than 4 logs of neuroblastoma cells were destroyed in the presence of a more than tenfold excess of bone marrow cells following two incubations with monoclonal antibody 6-19 and complement for 30 min at 37 degrees C. Monoclonal antibody concentrations greater than 5-10 micrograms/ml did not increase cell lysis. The destruction of tumor cells was limited by depletion of complement activity. Tumor cell killing increased with complement concentration and incubation duration but there was no additional cell lysis with incubations greater than 30 min. The percentage of target cells killed was significantly decreased when the target cells were treated in the presence of increasing concentrations of bone marrow mononuclear cells. This decrease in target cell destruction is a result of additional depletion of complement activity by bone marrow mononuclear cells. These results suggest that optimal purging of tumor cells by antibody and complement will be achieved following multiple brief incubations with fresh antibody and complement.     

An 80,000-kd glycoprotein cell surface antigen found only on nonhematopoietic cells in human bone marrow.

The murine IgG2a monoclonal antibody (MoAb) 6-19 binds to the surface of human nonhematopoietic cells but not to hematopoietic cells. As previously described, it can be used with complement to selectively kill nonhematopoietic cells prior to culture of human bone marrow. It is now shown that the 6-19 MoAb recognizes a specific antigenic determinant of apparent molecular mass 80 kd, detected by western blotting on nonhematopoietic tumor cell lines, endothelial cells, and bone marrow stromal cells. Mouse L cells were transfected with human DNA, and the 6-19 monoclonal antibody selected cells that expressed an antigen with similar characteristics. The antigenic determinant is absent in hematopoietic tumor cell lines, peripheral blood cells, and bone marrow hematopoietic cells. Investigation of the biochemical nature of the antigen, using various enzymes, lectin-binding studies, and western blotting suggests that the 6-19 epitope is at least partially carbohydrate and that the protein has N-linked sugars but not O-linked. The affinity (Ka approximately 10(9) M-1) of MoAb 6-19 binding is similar in tumor cells and normal fibroblasts and endothelial cells. The identification of a specific antigenic determinant of 80 kd may help to discriminate between hematopoietic and nonhematopoietic cells in human bone marrow.     

Elimination of clonogenic Burkitt's lymphoma cells from human bone marrow using 4-hydroperoxycyclophosphamide in combination with monoclonal antibodies and complement

One requirement for autologous bone marrow transplantation is the selective removal of malignant cells from normal marrow precursors. Development of a clonogenic assay that detects elimination of up to 5 logs of Burkitt's lymphoma cells in the presence of a 20-fold excess of human bone marrow has permitted the evaluation of two different methods for the selective removal of malignant cells. Treatment with 4- hydroperoxycyclophosphamide (4-HC) (60 to 100 micrograms/mL) eliminated 2.0 to 3.5 logs of clonogenic cells. Antitumor activity depended upon the concentration of 4-HC and the length of incubation, but not upon the concentration of normal bone marrow cells. Comparable removal of clonogenic Burkitt's cells was achieved by treatment with rabbit complement (C') and a combination of J5 anti-common acute lymphoblastic leukemia antigen (J5 anti-CALLA), J2 anti-gp 26, and the B1 anti-B1 murine monoclonal antibodies. A combination of 4-HC and monoclonal antibodies proved slightly but significantly more effective than either single agent in eliminating clonogenic tumor cells. Although treatment with 4-HC markedly reduced granulocyte-macrophage colony-forming units- C (GM-CFU-C) content of human bone marrow, neither treatment with 4-HC nor treatment with monoclonal antibodies and C' eliminated precursor cells that could generate new GM-CFU-C after growth in continuous bone marrow cultures. Our data suggest that treatment with 4-HC in combination with multiple monoclonal antibody reagents could be a safe and effective method of eliminating clonogenic tumor cells from human bone marrow.     

In vitro detection of occult bone marrow metastases in patients with colorectal cancer hepatic metastases

PURPOSE: The purpose of this study was to assess the immunocytochemical status of bone marrow aspirates from patients with clinically isolated hepatic metastases to test the hypothesis that such findings would allow improved patient selection for liver-directed treatment. METHODS: All patients had biopsy-proven or presumed colorectal cancer metastatic to the liver and were scheduled for an operative procedure for hepatic resection or for hepatic artery catheter and chemotherapy pump implant. Immunocytochemical analysis of bone marrow aspirate smears was performed with a panel of monoclonal antibodies directed toward cytokeratins, Lewis Y antigen and A-33 colorectal epitopes. RESULTS: Data from 80 patients indicated that bone marrow reactivity was present in 9.5 percent of those with resectable hepatic metastases and in 34 percent of those not resected (P=0.03). No single monoclonal antibody or combination produced better discrimination. CONCLUSIONS: Presence or absence of presumed occult colorectal cancer cells in the bone marrow of patients with isolated hepatic metastases is biologically interesting, but not useful in selecting or altering patient management.Supported in part by the Ludwig Institute.     

Idiotypic cascades in cancer patients treated with monoclonal antibody CO17-1A.

We have previously shown that gastrointestinal cancer patients treated with monoclonal antibody CO17-1A (Ab1) developed anti-idiotypic antibodies (Ab2) to the Ab1. We now demonstrate that patients produce anti-anti-idiotypic antibodies (Ab3) to their autologous Ab2. Ab3 were demonstrated in culture supernatants of peripheral blood mononuclear cells from five Ab1-treated patients after stimulation of the cells with heterologous Ab2 that functionally mimicked the tumor antigen (Ag) defined by Ab1 and immunologically cross reacted with the patients' Ab2. Ab3 shared idiotopes with Ab1 and were Ab1-like in their binding specificities to tumor cells, Ag, and Ab2. Such antibodies were also elicited by stimulating cells with Ag. However, they were not produced by stimulating posttreatment mononuclear cells with control proteins or by stimulating pretreatment cells with either Ag or Ab2. Our results demonstrate idiotypic cascades in cancer patients treated with monoclonal antibody. Ag-specific Ab3 responses may underlie delayed clinical responses often observed in cancer patients treated with monoclonal antibodies of various specificities.     

Treatment of two patients with B cell lymphoma with monoclonal anti- idiotype antibodies

Mouse monoclonal anti-idiotype antibodies have been used to treat two patients with progressive advanced B cell non-Hodgkin's lymphoma. Transient falls in the level of circulating malignant cells and idiotypic immunoglobulin were produced, and free unbound monoclonal antibody was identified in the serum. Homing of the antibodies to tumor cells in the blood, bone marrow, ascites, and lymph nodes was demonstrated in both patients. Although large amounts of anti-idiotype antibody were given (3.8 g and 5.8 g), no toxic effects were seen, and no antibodies to the foreign mouse protein were made. There was no modulation of the antigen from the tumor cells and no indication of immunoselection. There was evidence of large-scale tumor cell destruction, but only a modest reduction in tumor size. The killing of the tumor cells was mediated by the reticuloendothelial system and not by complement.     

A monoclonal antibody to myelogenous leukemia: isolation and characterization

A purified antigen from human acute myelogenous leukemia (AML) cells has been used to produce a myelogenous leukemia-associated monoclonal antibody. In limited FACS-IV analyses the monoclonal antibody to leukemia (CAMAL-1) as well as a conventional rabbit antiserum have been used to positively identify AML or chronic granulocytic leukemia patient cell samples. Neither CAMAL-1 nor the rabbit antiserum bound appreciably to acute lymphocytic leukemia cells, normal bone marrow, or normal peripheral blood leukocytes. CAMAL-1 was shown to be specific for AML cell extracts in the ELISA and was successfully used as an immunoadsorbent for the purification of the AML antigen from cell extracts. No significant levels of equivalent antigen were found when cell extracts from normal cells, lymphocytic leukemia cells, and lymphoma cells were similarly absorbed. These findings indicate that CAMAL-1 shows considerable specificity for an antigen associated with cells from patients with myelogenous leukemia.     

High-dose chemotherapy with autologous peripheral blood stem cell transplantation support in a patient with breast cancer metastasis to bone marrow: immunocytochemical monitoring of cancer-cell contamination]

A 32-year-old woman who 1 year earlier underwent a right mastectomy for stage II breast cancer with the histology of invasive ductal carcinoma (scirrhus type) was admitted due to recurrent, metastatic breast cancer in January 1997. She presented multiple metastatic lesions in the skin, lymph nodes, bone, lungs, liver, and spleen, and her bone marrow was replaced almost entirely by tumor cells. The patient was sequentially treated with 5 courses of cyclophosphamide (CPA) and adriamycin (ADM) (CA); 2 courses of CPA, ADM, and 5-fluorouracil; 5 caurses of docetaxel hydrate; and 1 course of CA. After recovery of the normal bone marrow by standard-dose chemotherapies, peripheral blood stem cells (PBSC) were then collected after mobilization with G-CSF. The number of breast cancer cells in bone marrow and PBSC samples was determined by immunocytochemical staining with an anti-cytokeratin monoclonal antibody. The number of tumor cells in PBSC sample was within the level for non-metastatic breast cancer. Complete remission was obtained with high-dose chemotherapy consisting of CPA and Thio-TEPA, and supported by autologous PBSC transplantation.     

S-L 11.14: a monoclonal antibody recognizing neuroectodermal tumors with possible value for bone marrow purging before autograft

A new monoclonal antibody (S-L 11.14) raised against small cell lung cancer reacted with all but one neuroblastoma tumor cell sample tested and was relatively specific for such cells within the bone marrow. The ability of S-L 11.14 to eliminate neuroblastoma cells from the bone marrow with an immunomagnetic purging method was evaluated in an experimental model using the LAN.1 and SKNBE neuroblastoma cell lines as targets. Residual malignant cells after the purging procedure were quantified by the Hoechst staining method. The use of S-L 11.14 as a single reagent resulted in a 3-log elimination of malignant cells, a depletion equal to that obtained with a cocktail of five monoclonal antibodies currently used in clinical trials. The addition of the S-L 11.14 antibody to this cocktail did not enhance depletion.     

Detection of metastatic tumour cells in routine bone marrow smears by immuno-alkaline phosphatase labelling with monoclonal antibodies

The present study describes 11 cases (10 carcinomas, one rhabdomyosarcoma) in which immuno-alkaline phosphatase labelling with monoclonal antibodies was used to demonstrate metastatic cells in routine smears of aspirated bone marrow. Carcinoma cells were detected using antibodies against epithelial cytokeratins, milk fat globule membrane antigen and carcinoembryonic antigen, and rhabdomyosarcoma cells with monoclonal anti-desmin. In four of the carcinoma cases it had not been possible to identify malignant cells in routinely stained marrow smears, whilst the case of disseminated rhabdomyosarcoma had initially been diagnosed (and treated) as a case of acute lymphoblastic leukaemia. The anti-cytokeratin antibody was found to be the most valuable of the anti-epithelial reagents used, since it labelled malignant cells in all of the 10 cases of carcinoma and gave the strongest reactions. These results suggest that immunocytochemical labelling should be used in cases of suspected carcinoma whenever conventional examination of marrow smears yields negative results, and furthermore (as illustrated by the case of rhabdomyosarcoma) that the technique is of value for identifying the true nature of poorly differentiated neoplasms in bone marrow.     

Treatment of severe Epstein-Barr virus-induced polyclonal B-lymphocyte proliferation by anti-B-cell monoclonal antibodies. Two cases after HLA-mismatched bone marrow transplantation

We treated two children who developed Epstein-Barr virus-induced polyclonal B-cell proliferation after HLA-mismatched bone marrow transplantation for congenital immunodeficiency with two monoclonal anti-B-cell antibodies. Lymphoproliferative syndrome occurred between 50 and 60 days after bone marrow infusion, and was diagnosed by the presence of spontaneously growing B cells containing Epstein-Barr-nuclear antigen in the blood and bone marrow. The mouse monoclonal anti-B-cell antibodies used were a CD21-specific antibody recognizing the CR2 receptor on B cells (BL13, IgG1) and a CD24-specific antibody binding B cells at all steps of differentiation (ALB9 IgG1). Both antibodies were given intravenously (0.2 mg/kg/body weight.d for 10 days). All clinical and biological manifestations resolved within 3 weeks of treatment. Recurrence was not seen at 18- and 15-month follow-ups. T-cell function developed normally; B-cell function remained partially deficient in one patient 21 months after bone marrow transplantation. These results suggest that monoclonal anti-B-cell antibodies could be useful in controlling severe polyclonal lymphoproliferative syndrome in profoundly immunodeficient patients after bone marrow transplantation.     

Differences and relationships of thymidine phosphorylase expression in tumor-associated macrophages and cancer cells in squamous cell carcinoma of the esophagus

Thymidine phosphorylase (TP), which has been shown to be identical to platelet-derived endothelial cell growth factor, is expressed in tumor-associated macrophages (TAMs) as well as cancer cells. The aim of this study was to clarify the differences or relationships of TP expression in TAMs and cancer cells in esophageal squamous cell carcinoma (SCC). Tissues samples were taken from 56 patients with esophageal SCC after curative surgery. The expression of TP in TAMs or SCC cells was examined using a monoclonal antibody to TP (clone 654-1). Microvessels in SCC that stained positively for Factor VIII-related antigen were counted (microvessel density, MVD). Macrophages in SCC that stained positively for CD68 antigen were counted (monocytic count). Ki-67 antigen was immunostained with MIB-1, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling was performed, and Ki-67 labeling index (LI) and apoptotic index were calculated. The expression of TP in stromal cells and cancer cells was observed in 43 (76.8%) and 33 patients (58.9%), respectively. There were significant correlations between TP expression in stromal cells (TAMs) as well as in cancer cells and venous invasion, distant metastasis, or MVD. There was a correlation between TP expression in cancer cells and lymph node metastasis, and there were correlations between TP expression in TAMs and monocytic count or Ki-67 LI; however, there was no correlation between TP expression in TAMs and lymph node metastasis. On the other hand, in SCCs with TP expression in both TAMs and cancer cells, higher frequencies of venous invasion and distant metastasis, higher MVD and lower apoptotic index were observed than in other SCCs. The 5-year survival rate in patients with TP expression in both TAMs and cancer cells was poorer than that in patients with TP expression in neither TAMs and cancer cell. In conclusion, these results suggest that co-expression of TP in TAMs and cancer cells is strongly associated with angiogenic promotion and distant metastasis. However, other effects of TP, such as promotion of tumor growth and lymph node metastasis, may be different depending on whether these are expressed in TAMs or cancer cells in esophageal SCCs. Patients with coexpression of TP in TAMs and cancer cells may be associated with a poor prognosis.     

Detection of lymph nodes micrometastases in Dukes＇ A and B colorectal cancer using anti-cytokeratin antibodies AE1／AE3

AIM: To detect lymph nodes micrometastases and analyze its correlation with clinicopathological parameters in Dukes' A and B colorectal cancer patients. METHODS: One hundred and fourteen patients with colorectal cancer (Dukes' A 16; Dukes' B 98) undergoing curative operation without histological lymph nodes metastases were studied between 2001 and 2003. A total of 2 481 lymph nodes were analyzed using monoclonal cytokeratin antibody AE1/AE3 (DAKO, Carpinteria, CA) for immunohist-ochemistry. RESULTS: In total, 33 (29%) patients were positive for cancer cell by immunohistochemistry. In 31 (94%) patients of them positive nodes showed single tumor cell or small groups of tumor cells; and tumor deposits measuring 0.2 and 0.37 mm in diameter in another 2 (6%) patients. Micrometastases were mainly located in the subcapsular sinus or paracortical sinus. There was no correlation between the positive lymph nodes and gender, age, tumor site, tumor size, histological type, histological grade, invasion depth, Dukes' staging and microsatellite instability (P>0.05). CONCLUSION: Our findings suggest that immunohist-ochemical technique using monoclonal cytokeratin antibody AE1/AE3 may be a sensitive and reliable method for detecting lymph nodes micrometastases in Dukes' A and B colorectal cancer. The clinical significance of lymph nodes micrometastases is still not confirmed.